The coagulation system in mammals is made of a series of proteins including pro-enzymes and co-factors interacting in a cascade type of activation. Upon activation, pro-enzymes convert into enzymes that, in the presence of the specific co-factor, cleave the next component in the cascade.
Such system is usually divided in three phases: an initiation phase, an amplification phase and a propagation phase. The initiation phase is triggered by the enzymatic cleavage of FX and FIX by tissue factor, in the presence of FVIIa and calcium. Activated FX cleaves prothrombin into thrombin. In the amplification phase thrombin activates a number of factors such as FV, FVIII and FXI, which in turn activates FIX. The propagation phase then is made of a number of positive feedback mechanisms, which result in further cleavage of FX by activated FIX combined with its co-factor FVIIIa. FXa and FVa associate to cleave prothrombin into thrombin.
Human FVIII is a 330 kd glycoprotein made of three domains containing two types of internal homologies. The first domain consists in the triplication of a A segment showing +/−30% homology between each other (A1, A2, A3) and encompassing residues 1-329, 380-711, 1,649-2,091, respectively. Regions A1 and A2 constitute the heavy chain, while A3, separated by a region of 948 amino acid rich in glycosylation sites (B domain) is located at the amino-terminal end of the light chain. The second internal homology is found at the carboxy-terminal end of the molecule where there are two copies of a third type of domain (C1 and C2) containing approximately 150 aminoacids with 40% homology. The native FVIII molecule made of the different segment separated by specific acidic regions (A1-a1-A2-a2-B-a3-A3-C1-C2) is rapidly cleaved by enzyme before entering in the plasma as an heterodimer consisting of a heavy chain (A1 and A2 domains together with the B domain or truncated part of it) associated by divalent cation to a 80 kD light chain (a3-A3-C1-C2). To become active and play its function in tenase complex formation, circulating FVIII has to be cleaved by thrombin.
Haemophilia A is characterized by the lack or insufficient function of FVIII. Patients with severe haemophilia A (namely, less than 1% functional FVIII), are treated by administration of recombinant or plasma-derived FVIII as a replacement therapy. About 25% of hemophilia A patients under replacement therapy by FVIII infusion develop an immune response to FVIII. This is due to the fact that severe haemophilia A patients have had no opportunity to become tolerant to FVIII because of lack of exposure of FVIII to their immune system during gestation. Anti-FVIII antibodies can also be found in the context of some autoimmune diseases, or occasionally after pregnancy or surgery. Such antibodies, called inhibitors, reduce the rate of thrombin generation by the tenase complex and thereby inhibit the amplification loop of the coagulation cascade.
Inhibitor antibodies recognize a number of discrete epitopes on the FVIII molecule. By far the most frequently recognized epitopes are located within the C2 and A2 domains. Extensive characterization of the epitopes in the C2 domain has been achieved thanks to the analysis of the crystal structure of C2 and to the availability of a human monoclonal anti-C2 antibody, which allowed a full validation of the structural model of the C2 domain, as well as a precise mapping of the epitope at single aminoacid level (Jacquemin M G et al., 1998 Blood. July 15; 92(2):496-506). This knowledge has opened two lines of investigations, namely the design of new FVIII molecules with reduced interaction with inhibitor antibodies and the development of new therapeutic strategies aiming at preventing or suppressing the production of anti-C2 inhibitor antibodies.
A similar approach to elucidate antibody interactions with the A2 domain has up to now been hampered by the lack of suitable reagent. The prior art described human monoclonal IgM antibodies with low affinities for the FVIII, but no human monoclonal IgG antibodies with strong FVIII inhibiting capacities have been described.
Antibodies derived from the repertoire of patients with inhibitors are unique reagents as they represent the actual antibodies generated towards FVIII. By contrast, antibodies raised in animal models such as the mouse are not representative of the human situation, as the characteristics of mouse immune system are not comparable to that of human.
Next to a potential use for characterization of FVIII or establishment of therapies aiming at preventing or suppressing the production of inhibitors, antibodies against the A2-domain of FVIII, whether or not derived from Hemophilia A patients, can also be useful for therapeutic purposes, e.g for inhibiting the formation of blood clots. Anticoagulation and antithrombotic treatment aim at inhibiting the formation of blood clots in order to prevent the dangerous consequences, such as myocardial infarction, stroke, loss of a limb by peripheral artery disease or pulmonary embolism. Until today, antithrombotic therapy relies on a few drugs since many years, namely Aspirin, heparin and oral Warfarin. With growing understanding of the processes involved in thrombosis a growing number of specific inhibitors of coagulation factors have been developed, such as recombinant tissue plasminogen activator (t-PA) or streptokinase. However, a better efficacy/safety ratio could to date not be obtained with them.
Monoclonal antibodies have already been shown to be of therapeutic value as antithrombotic agents. The first approved drug in this field was Abciximab (ReoPro™), a humanized Fab fragment of a murine monoclonal antibody (7E3) against platelet GP IIbIIIa receptors. Murine antibodies have characteristics which may severely limit their use in human therapy, since they may elicit an anti-immunoglobulin response termed human anti-mouse antibody (HAMA) that reduces or destroys their therapeutic efficacy and/or provokes allergic or hypersensitivity reactions in patients. While the use of human monoclonal antibodies would address this limitation, it has proven difficult to generate large amounts of such antibodies by conventional hybridoma technology.
Recombinant technology has therefore been used to construct “humanized” antibodies that maintain the high binding affinity of murine monoclonal antibodies but exhibit reduced immunogenicity in humans. Problems with binding affinity and side-effects like bleeding have been reported for several “humanized” antibody therapies.
Accordingly, novel anticoagulation and antithrombotic/thrombolytic treatments or in general compounds for the treatment of coagulation disorders are needed. For a therapeutic agent based on antibodies, the ideal compound is a human antibody with full anticoagulant efficacy that does not induce immunogenicity.
The prior art describes isolated human antibodies to the A2-domain of Factor VIII, obtained from lymphoblastoid cell lines producing anti-FVIII antibodies from peripheral blood mononuclear cells (PBMCs) of hemophilia A patients by EBV-immortalization (Gharagozlou et al. 2003, Human antibodies 12:67-76). All of the antibodies described are however exclusively of the IgM isotype and therefore difficult to use for therapeutic purposes. The authors furthermore suggest that there is a system of preferential expansion of the FactorVIII-specific IgM+ B-cells in hemophiliac patients which could be specifically associated with the properties of FVIII molecules and the conditions of the sensitization to FVIII in hemophilia patients, hereby suggesting that obtaining IgG antibodies through this way is not possible or extremely difficult.
Accordingly, there remains a need for monoclonal antibodies and antibody fragments, which bind to the A2 domain of Factor VIII and inhibit FVIII activity. Ideally, for use as therapeutic agents, such antibodies are non-immunogenic, in that they can not elicit HAMA (or have a low tendency to do so).